Comment[ArrayExpressAccession] E-GEOD-45183 MAGE-TAB Version 1.1 Public Release Date 2013-04-23 Investigation Title A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (expression between lines at timepoints). Comment[Submitted Name] A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (expression between lines at timepoints). Experiment Description A network model has been generated that describes the immediate gene expression cascade surrounding three similar but distinct NAC transcription factors that have roles to play in leaf senescence and in many stress responses in Arabidopsis. ANAC019, ANAC055 and ANAC072 belong to the same clade of NAC domain genes and have overlapping expression patterns. A combination of promoter DNA/protein interactions identified using yeast 1 hybrid analysis and modeling using gene expression time course data has been used to predict the regulatory network upstream of these genes. Similarities and divergence in regulation during a variety of stress responses are predicted by different combinations of upstream transcription factors binding and also by the modeling. Mutant analysis with potential upstream genes was used to test and confirm some of the predicted interactions.. Gene expression measurements in mutants of ANAC019 and ANAC055 at different times during leaf senescence have shown a distinctly different role for each of these genes. Leaf 7 from the anac019 (genotype 9C11) and anac055 (genotype IM4) mutants and their WT controls, Col-5 and Col-0 respectively, was tagged with cotton 18 days after sowing (DAS) and harvested from five randomly selected plants (biological replicates), 8 h into the light period, at 23, 29, 31, 33 and 35 DAS (full senescence). Variance in expression between lines at timepoints was analysed using a local adaptation of the MAANOVA package as previously described (Breeze et al., Plant Cell 2011 Mar;23(3):873-94. PMID: 21447789) using a loop design including dye swaps. Genotype DM (double mutant cross between IM4 and 9C11) was also included in the experiment, and was part of the loop design. Results for DM are included here in so far as they played a role in the ANOVA definition of values, but are not discussed in the paper. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Moore Hickman Hill Penfold Breeze Bowden Zhang Jackson Cooke Bewicke-Copley Moore Mead Beynon Wild Denby Ott Buchanan-Wollaston Person First Name Jonathan Richard Claire Christopher Emily Laura Peijun Alison Emma Findlay Jonathan Andrew Jim David Katherine Sascha Vicky Person Mid Initials David A D L Person Email jonathan.moore@warwick.ac.uk Person Affiliation University of Warwick Person Address Systems Biology, University of Warwick, Senate House, Coventry, United Kingdom Person Roles submitter Protocol Name P-GSE45183-1 P-GSE45183-5 P-GSE45183-6 P-GSE45183-2 P-GSE45183-3 P-GSE45183-4 P-GSE45183-7 Protocol Description Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.). Quantified microarray data were Lowess normalied within pintip groups and within arrays, then variation due to arrays and dyes was removed by a random effects model, and averaged, in log space, using R/MAANOVA (MicroArray ANalysis Of VAriance) (Wu et al. 2003). ID_REF = VALUE = Lowess normalized log2 signals Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]). The microarray experiments were carried out using the CATMA (version 4) microarray (Allemeersh et al., 2005; http://www.catma.org). Labelled samples were hybridized to slides overnight at 42oC. Leaf 7 was tagged with cotton 18 days after sowing (DAS) and harvested from five randomly selected plants, 8 h into the light period, at 23, 29, 31, 33 and 35 DAS (full senescence). Arabidopsis plants were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The anac055 line (genotype IM4, col0 background) was T-DNA insertion line Salk_011069 (obtained from the Nottingham Arabidopsis Seed Centre). The anac019 dSpm insertion mutant (genotype 9C11, col5 background) was identified in a pool of SLAT line DNA screened with gene specific primers (Tissier et al., 1999). Total RNA was isolated from four individual leaves from each sampled time point (arbitrarily labelled as biological replicates A, B, C and D) using TRIzol reagent (Invitrogen), purified with RNeasy columns (Qiagen) and amplified using the MessageAmp II aRNA Amplification kit (Ambion) in accordance with the kit protocol with a single round of amplification. Following hybridization, slides were washed and scanned using an Affymetrix 428 array scanner at 532nm (Cy3) and 635nm (Cy5). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name DAYS_AFTER_SENESCENCE GENOTYPE Experimental Factor Type days_after_senescence genotype Publication Title A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves. Publication Author List Hickman R, Hill C, Penfold CA, Breeze E, Bowden L, Moore JD, Zhang P, Jackson A, Cooke E, Bewicke-Copley F, Mead A, Beynon J, Wild DL, Denby KJ, Ott S, Buchanan-Wollaston V PubMed ID 23578292 Publication DOI 10.1111/tpj.12194 Comment[SecondaryAccession] GSE45183 Comment[GEOReleaseDate] 2013-04-23 Comment[ArrayExpressSubmissionDate] 2013-03-14 Comment[GEOLastUpdateDate] 2013-04-25 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-45183.sdrf.txt